Alfacalcidol restores cancellous bone in ovariectomized rats

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1 J Musculoskel Neuron Interct 23; 3(1):39-46 Originl Article Hylonome Alfclcidol restores cncellous one in ovriectomized rts M. Li, Y. Li, D.R. Hely, H.A. Simmons, H.Z. Ke, D.D. Thompson Osteoporosis Reserch, Deprtment of Crdiovsculr nd Metolic Diseses, Pfizer Glol Reserch nd Development, Groton, CT, USA Astrct Active vitmin D metolites hve een demonstrted to reduce verterl nd hip frctures in elderly ptients. A numer of in vitro nd in vivo pre-clinicl studies hve suggested tht vitmin D my effectively stimulte osteolstic ctivity nd exert n nolic effect on one. The current study ws designed to further explore the ility of n ctive vitmin D nlog to restore one in skeletl site with estlished osteopeni in ovriectomized () rts. Femle Sprgue Dwley rts t five months of ge nd 8 weeks fter shm ovriectomy or ovriectomy were rndomly divided into 7 groups with 1 per group. At the eginning of the tretments, one group of shm-operted rts nd one group of rts were scrificed to serve s seline controls. Another group of shm-operted rts nd one group of rts were treted with vehicle for 4 weeks. The rts in the remining groups were treted with lfclcidol t.5,.1 or.2 Ìg/kg/d y dily orl gvge, 5 dys/week for 4 weeks. As expected, estrogen depletion cused high one turnover nd cncellous one loss in lumr verter of rts. Alfclcidol tretment t.1 or.2 ut not.5 Ìg/kg/d incresed serum clcium nd phosphorus in rts s compred with vehicle tretment. In ddition, serum prthyroid hormone ws suppressed, wheres serum osteoclcin ws incresed y lfclcidol t ll dose levels. Furthermore, histomorphometric dt of 2nd lumr verterl ody reveled tht cncellous one volume in rts treted with lfclcidol t.1 or.2 Ìg/kg/d ws incresed to the level of shm-operted rts treted with vehicle. This increment in cncellous one mss ws ccompnied y increses in treculr numer nd thickness nd decrese in treculr seprtion. Moreover, osteoclst surfce nd numer were significntly decresed, wheres one formtion vriles such s minerlizing surfce nd one formtion rte were significntly incresed in lfclcidol-treted rts compred with those of vehicle-treted rts. Finlly, liner regression nlysis showed tht lfclcidol tretment dose-dependently ltered most of the vriles mesured in the current study. In conclusion, lfclcidol completely restores cncellous one y stimulting one formtion nd suppressing one resorption in lumr verter of rts when the tretment is strted t n erly phse of osteopeni. The evidence of incresed one formtion y lfclcidol tretments further supports the notion tht ctive vitmin D metolites or their nlogs my exert nolic effects on one. Keywords: Alfclcidol,, Cncellous Bone, Bone Resorption, Bone Formtion Introduction The ctive nd hormone form of vitmin D, 1,25-dihydroxyvitmin D 3 [1,25(OH) 2 D 3 or clcitriol] nd its pro-drug, 1 -hydroxyvitmin D 3 [1 (OH)D 3 or lfclcidol], hve een shown to reduce frcture rtes in postmenopusl nd senile osteoporosis, s well s glucocorticoid-induced osteoporosis 1-5. However, they hve not een pproved for the tretment of osteoporosis in the United Sttes due to the Corresponding uthor: Dr. Mei Li, Pfizer Glol Reserch nd Development, Deprtment of Metolic Diseses, MS8118W-28, Groton, CT 634, USA E-mil: mei_li@groton.pfizer.com Accepted 16 Octoer 22 risk of developing hyperclcemi/hyperclciuri, resulting in reltively nrrow therpeutic window 6. Therefore, it is importnt to understnd the mechnism of ction of vitmin D on one nd develop effective vitmin D nlogs without or with miniml clcemic effect. In vitro nd in vivo studies hve shown tht clcitriol hs stimultory effects on osteoclstogenesis nd ctivity 7-9. This effect is elieved to e medited through osteolstic cells 1,11, which possess undnt vitmin D receptors. However, the effects of clcitriol on one resorption re countercted in vivo y suppression of prthyroid hormone (PTH) secretion through oth direct inhiitory effect on the prthyroid glnds nd n indirect effect vi stimultion of intestinl clcium sorption nd susequent rise in serum clcium

2 M. Li et l.: Alfclcidol restores one in rts 4 8 Body Weights (g) A Shm Figure 1A Shm D3 (.5) D3 (.1) Serum Osteoclcin (ng/ml) E Shm Figure 1E Shm D3 (.5) D3 (.1) Serum Clcium (mg/dl) B Serum Phosphorus (mg/dl) C Serum PTH (pg/ml) D Shm Shm Shm Figure 1B Figure 1C Figure 1D Shm Shm Shm D3 (.5) D3 (.1) D3 (.5) D3 (.1) D3 (.5) D3 (.1) Figure 1. Body weights (A), serum clcium (B), serum phosphorus (C), serum PTH (D), nd serum osteoclcin (E) of ll groups. Dt re expressed s men ± SEM. p <.5 vs. Shm; p <.5 vs. Shm ; p <.5 vs.. More importntly, findings from previous in vitro nd in vivo studies hve suggested tht the vitmin D my effectively stimulte osteolstic ctivity nd exert n nolic effect on one. In vitro studies with osteolst model systems demonstrte tht clcitriol stimultes the synthesis of vriety of noncollgenous proteins such s osteoclcin, mtrix Gl protein, osteopontin, fironectin, s well s lkline phosphtse 12,13. In intct rts, high doses of clcitriol upregulte tiil osteoclcin messenger RNA levels 14 nd increse the numer of osteolst precursor cells in one mrrow of tii s well s stimulte cncellous one formtion in lumr verter 15. Previous studies using the ovriectomized () rt s model of postmenopusl one loss hve shown tht long-term tretments with clcitriol or lfclcidol increse one mss nd completely prevent one loss induced y estrogen deficiency However, s potentil nolic gent for the mngement of osteoporosis, it is importnt to know if ctive vitmin D metolites re cple of reuilding one mss in skeleton with estlished osteopeni. Preclinicl studies designed to ddress this issue re very limited nd the effects of ctive vitmin D metolites on one formtion oserved in those studies were not consistent 26,27. Therefore, the current study ws designed to further test the efficcy of ctive vitmin D metolites in restoring one mss to n osteopenic skeleton y using the rt s model nd lfclcidol s n gent. Mterils nd methods Animls nd Experimentl Design. Femle Sprgue Dwley rts t five months of ge nd 6 weeks fter shm ovriectomy or ovriectomy were purchsed from Tconic Frms Inc. (Germntown, NY). They were cclimted t 4

3 M. Li et l.: Alfclcidol restores one in rts Vriles BW C Pi PTH Osteoclcin R p.5 < <.1 BW: ody weight; C: serum clcium; Pi: serum phosphorus; PTH: serum prthyroid hormone; osteoclcin: serum osteoclcin. Tle 1. Dose response for non-histomorphometric vriles determined y liner regression nlysis. 24 O C with 12h light/12h drk cycle for 2 weeks efore initition of the study. They were llowed free ccess to wter nd commercil diet (Purin Lortory Rodent Chow 51, Purin-Mills, St. Louis, MO) contining.95% clcium,.67% phosphorus, nd 4.5 IU/g vitmin D 3. The experiment ws conducted ccording to Pfizer, Inc. niml crepproved protocols, nd nimls were mintined in ccordnce with the ILAR (Institute of Lortory Animl Reserch) Guide for the Cre nd Use of Lortory Animls. At 8 weeks post-surgery, the nimls weighing n verge of 38g were rndomly divided into 7 groups with 1 per group. One group of shm-operted rts ( Shm) nd one group of rts ( ) were scrificed to serve s seline controls. Another group of shm-operted rts nd one group of rts were treted with vehicle (Shm nd ) for 4 weeks. The rts in the remining groups were treted with.5,.1 or.2 Ìg/kg/d ody weight of lfclcidol (Sigm, St. Louis, MO) y dily orl gvge t volume of 1ml/kg ody weight, 5 dys/week for 4 weeks. Thus, this study ws comprised of the following dditionl tretment groups: D 3 (.5), D 3 (.1), nd D 3 (.2). The stock solution ws mde y dissolving lfclcidol in 1% ethnol t concentrtion of.1 mg/ml, protected from light, nd stored t 4 O C. The dosing solutions were prepred weekly y diluting the stock solution with cottonseed oil vehicle to given concentrtion. All rts were sucutneously injected with clcein t dose of 1 mg/kg (Sigm Chemicl Co., St. Louis, MO) oth -12 nd -2 dys prior to scrifice. This regimen resulted in deposition of single or doule fluorochrome lel t one surfces tht were ctively minerlizing t the time of the injections. At the conclusion of 4 weeks of tretment with lfclcidol, the nimls were fsted 16 hours prior to necropsy. At the dy of necropsy, 3 Ìl of lood ws first collected from ech rt y til vein leeding for the serum prthyroid hormone (PTH) level. The whole lood smples were then collected y crdic puncture for iochemicl ssessments right fter scrifice y CO 2 sphyxition. Blood smples were further centrifuged to otin the ser, which were stored t 2 O C until ssy. Finlly, 2 nd lumr vertere (LV2) were collected for one histomorphometric nlysis. Serum Biochemistry. Serum clcium (C) nd inorgnic phosphorus (Pi) concentrtions were mesured with the Cos Fr 2 nlyzer (Roche Dignostic System, Hoffmn- L Roche Inc., Indinpolis, IN). The serum PTH levels were mesured using rt PTH ELISA kit with detection limit t 1.6 pg/ml (Immutopics, Inc., Sn Clemente, CA) nd the serum osteoclcin concentrtions were mesured with rt osteoclcin ELISA kit (Biomedicl Technologies, Inc., Stoughton, MA). Cncellous Bone Histomorphometry. The LV2 were dehydrted in grded concentrtions of ethnol nd emedded undeclcified in methyl methcrylte 28. Longitudinl frontl sections of lumr verter were cut t 4- nd 1-Ìm thickness using Reichert-Jung Polycut S microtome (Cmridge Instruments, Heidelerg, Germny). The 4-Ìm sections were stined with modified Msson s Trichrome stin, nd the 1-Ìm sections remined unstined for mesurements of fluorochrome-sed indices of one formtion. All histomorphometric mesurements were performed in cncellous one tissue of the lumr verterl ody in n re etween.5 mm distl to the crnil nd.5 mm proximl to the cudl growth pltes using n Imge Anlysis System (Osteomesure, Inc., Altnt, GA). Cncellous one volume s percentge of one tissue re (BV/TV) nd osteoclst surfces s percentges of totl cncellous perimeter (Oc.S/BS) were mesured in 4-Ìm thick, stined sections. Treculr numer (T.N), width (T.Th) nd seprtion (T.Sp) were clculted s descried y Prfitt et l. 29. Fluorochrome-sed indices of one formtion including the percentge of cncellous one surfce with doule fluorochrome lel (minerlizing surfce, MS/BS) nd minerl pposition rte (MAR) were mesured in 1-Ìm thick, unstined sections. In ddition, one formtion rte (one surfce referent, BFR/BS) ws clculted y multiplying minerlizing surfce y minerl pposition rte. Vlues for minerl pposition rte were not corrected for oliquity of the plne of section in cncellous one 3. Sttisticl nlysis. Dt re expressed s the men ±SEM for ech group. Sttistics were clculted using SttView 4. pckges (Acus Concepts, Inc., Berkeley, CA). Sttisticl differences etween groups were evluted with ANOVA followed y the Fisher PLSD test for multiple comprisons. Proilities (p) less thn.5 were considered significnt. Dose-dependent responses of lfclcidol were determined y liner regression nlysis using SttView 4. pckges. 41

4 M. Li et l.: Alfclcidol restores one in rts Vriles BV/TV T.N T.Th T.Sp Oc.S/BS MS/BS MAR BFR/BS R p <.1.26 <.1.8 <.1 < BV/TV: cncellous one volume; T.N: treculr numer; T.Th: treculr thickness; T.Sp: treculr seprtion; Oc.S/BS: osteoclst surfce; MS/BS: minerlizing surfce; MAR: minerl pposition rte; BFR/BS: surfce-sed one formtion rte. Tle 2. Dose response for histomorphometric vriles determined y liner regression nlysis. Results Effects of ovriectomy in seline nd vehicle-treted rts. Bseline nd vehicle-treted rts weighed significntly more thn seline shm controls nd vehicletreted shm controls, respectively (Figure 1A). Serum clcium (Figure 1B), phosphorus (Figure 1C), nd PTH (Figure 1D) of rts were not different from shmoperted controls. The men vlue for serum osteoclcin (Figure 1E) in rts ws incresed t 2 months nd ck to the shm control level t 3 months post-. As expected, cused significnt one loss in the lumr verter of rts s compred with shm controls (Figure 2A). The reduction in verterl cncellous one mss in rts ws ccompnied with decrese in treculr numer (Figure 2B) nd increse in treculr seprtion (Figure 2D). Treculr thickness ws not different etween nd shm-operted rts (Figure 2C). Bone resorption nd one formtion vriles including osteoclst surfce (Figure 2E), minerlizing surfce (Figure 2F), minerl pposition rte (Figure 2G), nd one formtion rte (Figure 2H) were elevted in seline rts compred with seline shm nimls. At the end of the experiment, minerlizing surfce nd one formtion rte ut not osteoclst surfce nd minerl pposition rte in vehicle-treted rts remined higher thn those for vehicle-treted shm controls. Effects of lfclcidol in rts. The men ody weight of rts treted with the highest dose of lfclcidol ws decresed y 11% compred with tht of vehicle-treted rts (Figure 1A). Tretment of rts with.1 or.2 Ìg/kg/d of lfclcidol incresed serum C (8% nd 18%, respectively, Figure 1B) nd Pi (24% nd 45%, respectively, Figure 1C) significntly reltive to vehicle tretment. Serum PTH ws decresed y 91% in the rts treted with the lowest dose of lfclcidol nd it ws decresed to undetectle levels y the two higher doses of lfclcidol used in this study (Figure 1D). Tretment of rts with lfclcidol t ll dose levels elevted serum osteoclcin y 47% to 112% reltive to vehicle tretment (Figure 1E). Cncellous one histomorphometric dt from LV2 (Figures 2A to H nd Figure 3) showed tht lfclcidol t.1 nd.2 ut not.5 Ìg/kg/d significntly incresed cncellous one volume y 33% nd 46%, respectively, s compred with vehicle tretment in rts (Figure 2A). At the end of the 4-week tretment with lfclcidol t these dose levels, the men vlues of cncellous one volume in rts were equivlent to those in vehicle-treted shm controls. The increses in verterl cncellous one mss in rts were ccompnied y structurl improvement of treculr one, s reveled y increses in treculr numer (Figure 2B) nd thickness (Figure 2C), nd decrese in treculr seprtion (Figure 2D). In ddition, osteoclst surfce (Figure 2E) ws significntly decresed y lfclcidol tretments t ll dose levels. In contrst to osteoclst surfce, minerlizing surfce (Figures 2F nd 3) ws significntly incresed in rts treted with.1 nd.2 Ìg/kg/d of lfclcidol y 25% nd 52%, respectively, compred with tht of rts treted with vehicle. Furthermore, minerl pposition rte (Figure 2G) ws not ltered y lfclcidol tretment except it ws decresed in rts treted with the lowest dose of the hormone when compred with vehicle-treted rts. Finlly, one formtion rte (Figure 2H) ws incresed in rts treted with lfclcidol t.1 nd.2ìg/kg/d dose levels y 14% nd 45%, respectively, wheres t the.5 Ìg/kg/d dose level, lfclcidol decresed one formtion rte in rts s compred with vehicle. A liner regression nlysis showed tht lfclcidol tretment dose-dependently ltered most of the vriles mesured in the current study. There ws dose-dependent decrese in ody weight, serum PTH, treculr seprtion, nd osteoclst surfce in rts treted with lfclcidol. In contrst, lfclcidol tretment incresed the following prmeters in dose-dependent mnner: serum C, Pi, osteoclcin, cncellous one volume, treculr thickness, minerlizing surfce, nd one formtion rte (Tles 1 nd 2). Discussion The current study demonstrtes tht lfclcidol dosedependently restores cncellous one in the verter of osteopenic, rts. The restortion of cncellous one ws ccompnied with n improvement in treculr rchitecture s evidenced y incresed treculr numer nd thickness nd decresed treculr seprtion. In ccordnce with previous studies with ctive vitmin D metolites 16-27, the current study showed tht lfclcidol dosedependently suppressed one resorption in rts. This 42

5 M. Li et l.: Alfclcidol restores one in rts BV/TV (%) Oc.S/BS (%) 2 1 A Shm Shm D3 (.5) D3 (.1) E Shm Shm D3 (.5) D3 (.1) Figure 2A Figure 2E T.N (#/mm) MS/BS (%) B Shm Shm D3 (.5) D3 (.1) F Shm Shm D3 (.5) D3 (.1) Figure 2B Figure 2F T.Th (Ìm) 1 5 MAR (Ìm/d) 1.5 C Shm Shm D3 (.5) D3 (.1) G Shm Shm D3 (.5) D3 (.1) Figure 2C Figure 2G 3 T.Sp (Ìm) D 2 1 Shm Figure 2D Shm D3 (.5) D3 (.1) BFR/BS (Ìm 2 /Ìm/d) H Shm Figure 2H Shm D3 (.5) D3 (.1) Figure 2. Cncellous one volume (A), treculr numer (B), treculr thickness (C), treculr seprtion (D), osteoclst surfce (E), minerlizing surfce (F), minerl pposition rte (G), nd one formtion rte (H) of second lumr verter. Dt re expressed s men ± SEM. p <.5 vs. Shm; p <.5 vs. Shm ; p <.5 vs.. 43

6 M. Li et l.: Alfclcidol restores one in rts Figure 3. Cncellous one tissue in the 2nd lumr verterl ody of shm-operted rt treted with vehicle (A), n rt treted with vehicle (B), nd n rt treted with lfclcidol t.2 Ìg/kg/d (C). Note the decresed cncellous one mss in the rt treted with vehicle. Alfclcidol tretment completely restored cncellous one nd incresed minerlizing surfce (rrows) in the rt. Originl mgnifiction x125. inhiitory effect of lfclcidol on one resorption ws ccompnied with dose-dependent increse in serum clcium levels nd decrese in serum PTH levels in rts. The oserved hyperclcemi is proly due to incresed intestinl sorption of clcium y lfclcidol tretments. Since PTH is n importnt physiologicl regultor of one resorption nd one turnover, the suppression of one resorption y lfclcidol in the current study is considered to e lrgely consequence of diminished PTH ctivity. Although n cute increse in serum clcium leds to the secretion of clcitonin, which in turn inhiits osteoclst ctivity 31, studies hve shown tht long-term tretment of rts with clcitriol decreses clcitonin iosynthesis nd secretion 32,33. Therefore, it is unlikely tht clcitonin contriuted to the decresed one resorption fter lfclcidol tretment in the rts. Despite the sustntil suppression of PTH secretion nd one resorption, lfclcidol t the lowest dose employed in this study filed to significntly increse one in rts. This filure my e ttriutle to the decreses in minerl pposition rte nd one formtion rte oserved in these nimls. The decrese in one formtion my e coupling phenomenon ssocited with decresed one resorption y lfclcidol tretment t this dose level. Similr results hve een reported in rts fter 3 months of tretment with lfclcidol strting 4 dys or 2 weeks fter surgery 18,34. In contrst to the lowest dose, the two higher doses of lfclcidol pplied in this study restored verterl cncellous one mss to the control level in rts. Despite the sustntil decrese in serum PTH, one formtion indexes such s serum osteoclcin, one formtion rte, nd in prticulr minerlizing surfce were significntly incresed in these nimls. These results suggest tht high doses of lfclcidol stimulte one formtion in the rts. Therefore, the comined effects of lfclcidol t.1 nd.2 Ìg/kg/d on stimulting one formtion nd inhiiting one resorption resulted in the restortion of one in the rts in this study. The increse in minerlizing surfce nd one formtion rte y 4 weeks of tretment with lfclcidol shown in this experiment is consistent with the results from studies in which the rts received only short-term tretment with clcitriol 15,35. In those studies, percent treculr one surfce covered y mture osteolst cells ws incresed following 3 or 13 dys of clcitriol tretments. Increse in serum mrkers of one formtion such s osteoclcin 36,37 nd procollgen type I C-terminl propeptide (PICP) 37 hs lso een reported in humns hving received 4- or 7-dy tretments with clcitriol. Interestingly, the incresed one formtion y lfclcidol in this study does not gree with the dt from studies in which the nimls received long-term (12 weeks) tretment with clcitriol or lfclcidol In those long-term studies, one formtion prmeters generted y histomorphometric mesurements in the rts treted with clcitriol or lfclcidol were not different or lower 26 thn those in the rts treted with vehicle t the end of the 12-week tretment period. Tken together, ctive vitmin D metolite, clcitriol, my trnsiently stimulte one formtion during the initil phse of tretment. Such incresed one formtion y ctive vitmin D metolites my e overridden y the suppression of one formtion coupled with the inhiition of one resorption during the long-term tretment period. The study clerly showed tht lfclcidol t higher doses (.1 nd.2 Ìg/kg/d) completely restored cncellous one to the lumr verter of rts. This finding is somewht superior to the previous findings in rts treted with clcitriol. A study y Eren et l. 27 showed tht 3 months of tretment with clcitriol only prtilly restores cncellous one in, osteopenic rts. When compring the current study with Eren s study, it is notle tht the mgnitude of one 44

7 M. Li et l.: Alfclcidol restores one in rts lost in the lumr verter of rts t the eginning of the tretments is different, 19% decrese for the current study (2 months post-) nd 37% decrese (3 months post-) in Eren s study. The prtil restortion t the sme one site y clcitriol in Eren s study is most likely due to the greter one loss t the strt of the tretment, lthough other fctors such s the ge of the rts nd phrmcokinetics of pro-drug vs. ctive hormone cnnot e excluded. The even weker effect seen in the proximl tii, one site tht hd lost 74% of its cncellous one s compred with 37% in the lumr verter t the eginning of the tretment in the sme study 27 further supports the forementioned ssumption. A similr phenomenon hs een seen in the, osteopenic rts treted with PTH, i.e., PTH filed to completely restore cncellous one to skeletl site with severe one loss 38. The oservtions in the current study, in concert with previous findings, suggest tht in order to chieve the mximl efficcy, tretment with clcitriol or lfclcidol needs to e initited t n erly stge of one loss. However, the mximum effect of clcitriol or lfclcidol my not hve een estlished due to the inility to expnd the dose of the compounds ecuse of dverse effects. Therefore, it is not known if n effective vitmin D nlog, without ffecting clcium homeostsis, would chieve superior efficcy in restoring one to the osteopenic skeleton. Nevertheless, the incresed one formtion nd incresed one mss y lfclcidol tretments in rts, s demonstrted in our study, furthers the notion tht ctive vitmin D metolites or its nlogs my exert n nolic effect on one y directly up-regulting osteolstic cells. In greement with previous reports 18,19,21, lfclcidol t ll dose levels tested in the current study cused hyperclcemi in rts under clcium nd vitmin D-replete condition. This undesirle effect hs complicted the understnding of the direct effects of lfclcidol on one resorption nd formtion in vivo, nd limited the usge of these gents for the prevention nd tretment of osteoporosis. In recent yers, efforts hve focused on identifying potent nd tissue selective vitmin D nlogs to ddress this issue. Ro , vitmin D nlog, hs een recently reported to decrese urinry pyridinoline excretion nd increse osteolst numer s well s one mss in rts t doses tht hve no significnt effects on serum nd urine clcium levels. In ddition, the osteolst-specific gene products osteoclcin nd osteopontin s well s one growth fctors TGF 1 nd 2 mrna were up-regulted in treculr one y Ro These findings suggest the possiility of developing one-selective vitmin D nlogs with wider rnge of therpeutic window. In conclusion, lfclcidol completely restores cncellous one y stimulting one formtion nd indirectly suppressing one resorption in lumr verter of rts when the tretment is strted t n erly phse of one loss. The findings of incresed one formtion nd one mss y lfclcidol tretments further support the notion tht ctive vitmin D metolites or their nlogs my exert n nolic effect on one. References 1. Gllgher JC, Goldgr D. Tretment of postmenopusl osteoporosis with high doses of synthetic clcitriol. A rndomized controlled study. Ann Intern Med 199; 113: Tilyrd MW, Spers GFS, Thomson J, Dovey S. Tretment of postmenopusl osteoporosis with clcitriol or clcium. N Engl J Med 1992; 326: Sirnen S, Kärkkäinen M, Tähtelä R, Litinen K, Mäkelä P, Lmerg-Allrdt C, Välimäki MJ. Bone mss nd mrkers of one nd clcium metolism in postmenopusl women treted with 1,25-dihydroxyvitmin D (clcitriol) for four yers. Clcif Tissue Int 2; 67: Shirki M, Ito H, Orimo H. 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